RESUMEN
33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.